A central issue in developmental neurobiology is determining how neurons find their appropriate targets. Recently, it has become clear that neuronal growth cones respond to guidance cues that are both attractive and inhibitory, and that precise axon pathfinding, target recognition, and synapse formation result from a balance of these cues. The focus of the experiments described in this proposal is to elucidate the function during neurodevelopment of the Semaphorins: a family of growth cone guidance molecules, conserved from insects to mammals, which is implicated in inhibitory guidance. Understanding the molecular basis of inhibitory neuronal guidance is likely to be clinically important for therapeutic strategies directed toward addressing problems of nerve regeneration, neurodegeneration, and neuronal developmental disorders. To address how Semaphorins mediate neuronal growth cone guidance during development, we will utilize complementary approaches afforded by analysis in both invertebrates and vertebrates. To learn how Semaphorins function at the cellular level in vivo, we will analyze loss and gain-of- function mutations in the genes that encode Drosophila Semaphorins. We will investigate the function during neurodevelopment of discrete domains found in members of the Semaphorin gene family by undertaking a molecular dissection of insect and vertebrate Semaphorins. We will construct altered Semaphorins and test their function in vivo in Drosophila and in a vertebrate embryonic neuronal cell culture system. This analysis will also include a functional dissection of the phylogenetically conserved Semaphorin domain itself. To identify Semaphorin receptors and downstream signaling components, we will initiate genetic strategies in Drosophila. These studies will lead to an understanding at the molecular level of how Semaphorins help mediate the processes of axon pathfinding, target recognition, and synapse formation. These experiments should also identify phylogenetically conserved principles of Semaphorin function and should provide insight into the molecular mechanisms of inhibitory neuronal growth cone guidance.